The protective effect of vinpocetine against Estradiol-benzoate induced cervical hyperkeratosis in female rats via modulation of SIRT1/Nrf2, and NLRP3 inflammasome

The current study was assigned to determine the putative preventive role of vinpocetine (VIN) in cervical hyperkeratosis (CHK) in female rats. Estradiol Benzoate (EB) was utilized in a dose f (60 μg/100 g, i.m) three times/week for 4 weeks to induce cervical hyperkeratosis. VIN was administered alone in a dose of (10 mg/kg/day, orally) for 4 weeks and in the presence of EB. Levels of malondialdehyde (MDA), total nitrites (NOx), reduced glutathione (GSH), interleukin-18 (IL-18), IL-1β, tumor necrosis factor-alpha (TNF-α) were measured in cervical tissue. The expression of NLRP3/GSDMD/Caspase-1, and SIRT1/Nrf2 was determined using ELISA. Cervical histopathological examination was also done. EB significantly raised MDA, NOx, TNF-α, IL-18, IL-1β, and GSDMD and up-regulated NLRP3/Caspase-1 proteins. However, GSH, SIRT1, and Nrf2 levels were reduced in cervical tissue. VIN significantly alleviates all biochemical and histopathological abnormalities. VIN considerably mitigates EB-induced cervical hyperkeratosis via NLRP3-induced pyroptosis and SIRT1/Nrf2 signaling pathway.


Sample collection
At the close of the experiment, rats received an i.p. injection of urethane (25% in a dose of 1.6 g/kg) 35 .Rats were euthanized by cervical dislocation, and their cervices were removed and cleansed with saline to eliminate any blood.A portion was kept for histological examination.The other parts were split into two portions, the first of which was immediately frozen at -80°C until utilized for western blot analysis.For measuring biochemical parameters, the second portion was homogenised with phosphate buffer (0.01 M, pH 7.4; 20% w/v) (tissue weight (g): phosphate buffer (mL) volume = 1:5) 36 , then the homogenate was centrifuged for 15 min at 5000 rpm, and the supernatant was kept at -80 °C.

Biochemical investigations
Assessment of oxidative stress parameters Cervical reduced glutathione (GSH); (Cat.No.: GR 25 11), and malondialdehyde (MDA); (Cat.No.: MD 25  29) were measured by kits provided by Biodiagnositic, Giza, Egypt.Total nitrite/nitrate (NOx) was determined using the Griess reaction between nitrite and a mixture of naphthyl ethylenediamine and sulfanilamide; the NO level was detected at 540 nm 37 .

Histopathological study
The cervices of the female rats were removed, processed, and embedded in paraffin after being submerged in a 10% formalin solution for 24 h.With a microtome, cross-sections 5 μm thick were cut.Hematoxylin-eosin stain was applied to these tissue sections, and they were examined using an Olympus light microscope for a histological assessment.

Statistical analysis
The mean ± SEM was used to present all the data.The Tukey-Kramar test was conducted after a one-way analysis of variance (ANOVA) was used to examine the data.Significant P values were defined as those less than 0.05.The statistical analysis was carried out with GraphPad Prism ® , Version 10.00 for Windows.

Impact of VIN on oxidative stress parameters in cervical tissue
As presented in Table 1, EB significantly increased MDA and NO and reduced GSH, as compared to control group.On the other hand, VIN when co-administered with EB, significantly reduced both MDA and NOx and increased GSH, relative to EB groups.

Impact of VIN on cervical inflammatory markers
In group received EB, TNF-α, IL-18, and IL-1β were increased significantly, relative to control group.In contrast, in EB/VIN group, a significant reduction in TNF-α, IL-18, and IL-1β occurred, as compared to EB group (Fig. 1).

Impact of VIN on cervical SIRT1/Nrf2 signaling pathway
As presented in Fig. 2, EB significantly reduced SIRT1 and Nrf2, relative to control group.However, VIN when co-administered with EB, significantly reversed the condition as it increased both SIRT1 and Nrf2, as compared to EB group.

Impact of VIN on cervical NLRP3/GSDMD/Caspase 1 signaling pathway
In the EB group, NLRP3, GSDMD, and caspase-1 levels were all considerably increased, in relation to the control group.In contrast to EB group, In EB/VIN group, a significant decrease was observed in NLRP3, GSDMD, and caspase 1 (Fig. 3).

Histopathological results
Sections examined from control and VIN groups showed normal cervices lined by stratified squamous non keratinized epithelium.In contrast, cervices of group received EB showed marked and diffuse areas of hyperkeratosis.On the other hand, group in which VIN was co-administered with EB, only mild focal areas of hyperkeratosis were noticed (Fig. 4).

Discussion
CHK is a prevalent gynecological condition caused by excessive administration of estrogen 5 .Over-administration of estrogen contributes significantly to CHK and cervical cancer by inducing cytokines, oxidative stress, and the generation of free radicals 10 .Estrogen administration, particularly when combined with other substantial risk factors such as multiparity and human papillomavirus (HPV) infection, can lead to cervical cancer 38 .In the current study, EB used as a positive control drug that induced CHK as mentioned in previous studies and confirmed by a typical histopathological alterations 3,39 .The development of CHK is heavily influenced by oxidative stress 3 .We investigated oxidative stress markers such as MDA, NOX, and GSH to assess the oxidative stress effect of EB therapy.The negative impacts of EB were demonstrated by a considerable rise in cervical MDA, and NOx, as well as a significant decrease in cervical GSH relative to the control group.Co-administration of VIN with EB in the current study significantly improved oxidative stress status as shown by reduction in MDA, NOx and increase in GSH.This result indicates an antioxidant effect of vinpocetine in CHK.This is also documented by recent Fattori et al., study showing that vinpocetine has a considerable oxidative stress ameliorating effect 40 .
Earlier studies demonstrated that EB administration can trigger CHK by stimulating inflammation and releasing inflammatory mediators such as TNF-α, and IL1β 3,41,42 .Our study showed a significant rise in TNF-α, IL18, and IL1β levels in the EB group.Fortunately, VIN significantly reduced TNF-α, IL18, and IL1β levels, demonstrating its protective effect against EB-induced CHK, which is in line with previous studies that reported the antioxidant, and anti-inflammatory properties of VIN in several animal models, including acute kidney injury, lung inflammation caused by lipopolysaccharide, otitis media in mice, and inflammatory pain [43][44][45] .
To acquire a better understanding of the mechanism of VIN's protective impact against EB-induced CHK, NLRP3, Caspase-1, and GSDMD levels were investigated.NLRP3 (nucleotide-binding oligomerization domain receptors) are intracellular proteins that play a function in mammalian immunity and are highly expressed in cervical carcinoma 17 .To form an inflammasome complex, NLRP3 binds to ASC and subsequently activates procaspase 1. Mature IL-1β and IL-18 are produced from pro-IL-1β and pro-IL-18 by active caspase1, However, caspase1 also encourages GSDMD to become cleaved GSDMD, which causes the plasma membrane to open up significantly and starts the process of pyroptosis 46 .Our study showed a significant rise in cervical NLRP3, Caspase-1, and GSDMD levels in the EB group demonstrating that NLRP3 inflammasome is strongly associated with CHK.Conversely, VIN resulted in a markedly reduced expression of NLRP3, Caspase-1, and GSDMD.This is consistent with the findings of Dong Han et al. (2020), whereby the NLRP3 signaling pathway was suggested as a plausible explanation for VIN's ability to mitigate ischemic stroke 47 .
In the same vein, EB injection induced histological alterations, characterized by prominent hyperkeratosis with a thicker keratin layer on the surface of stratified squamous epithelium with underlying significant stromal inflammatory cell infiltration.These findings are in line with previous studies 48,49 .In EB/VIN group, there is marked improvement of these histopathological alterations.As only mild focal areas of hyperkeratosis were noticed.Additionally, the improvement of histopathological aberrations was supported by the downregulation of cervical inflammatory cytokines, NLRP3, caspase 1, and oxidative stress markers.
To provide more insight into the potential protective mechanism of VIN against EB-induced CHK, an evaluation of the SIRT1/Nrf2 signaling pathway was conducted.As many of its downstream target genes and enzymes are in charge of avoiding or reversing intracellular redox imbalances, Nrf2 is regarded as a master regulator of the antioxidant response 50,51 .Well-known stress response protein SIRT1 is essential for a variety of cellular and physiological processes including cell damage, and mitochondrial biogenesis and its expression is correlated with endometrial cancer 23,52 .Moreover, some investigations have suggested that SIRT1 may activate Nrf2 in order to exhibit its antioxidative actions 53,54 .Earlier studies demonstrated that downregulating Nrf2 is associated with NLRP3 activation and release of inflammatory mediators supporting the connection between the two investigated pathways in the current work 55,56 .According to the results of the current investigation, the SIRT1 and Nrf2 levels were lower in the EB-treated group.Inversely, VIN increased SIRT1 and Nrf2 expression.Additionally,

Conclusion
In the current study, collectively, VIN antagonized oxidative stress, and inflammation induced by EB as evidenced by reduction in MDA, NOx, TNF-α, IL18, and IL1β with increase in GSH and via stimulation of SIRT1/Nrf2 signaling pathway and subsequent inhibition of NLRP3/Caspase 1/ GSDMD signaling pathway as illustrated in Fig. 5.

Figure 5 .
Figure 5. Graph outlining the mechanism of EB-induced CHK and the potential protective effect of VIN.One of the authors, Ehab E. Sharata, used Microsoft PowerPoint to create this graph.